A key goal of retroviral-based gene delivery is to maximize gene expression without the complication of insertional mutagenesis. The studies in this proposal examine the linkage between integration and gene expression. This new area of investigation is based on the observation that mutations within the Moloney murine leukemia virus integrase (M-MuLV IN) proteins can be compensated by the presence of histone deacetylase (HDAC) inhibitors at the time of integration. The results highlight a new function of the IN protein beyond integration, namely the regulation and establishment of the epigenetic state of the proviral DNA. In a wildtype MuLV infection, viral expression is suppressed until after integration, which results in highly expressed gene products. Analysis of mutations within the MuLV IN C- terminal domain (CTD) reveals a phenotype that has lost this tight regulation. Although replication and integration of the K376R mutant is equivalent to WT IN, the expression from the integrated virus is minimal. Experiments aim at defining how the IN protein is involved in the early establishment of short-term gene expression from the provirus. Additional experiments aim at defining the normal mechanism of transcriptional repression from unintegrated DNA. The role of the IN protein and the intasome in repressing the LTR enhancer/promoter regions will be studied. The relationship of the promoter with respect to the viral termini will be examined. Additional studies aim at manipulating the expression from the unintegrated virus. The final specific aim provides structural/functional analysis of the IN CTD. Two collaborative projects have been established. The first examines the role of host proteins to interact with the MuLV IN CTD. The second is a NMR structural study of the MuLV IN CTD. These studies have direct application to multiple areas of vector development. The ability to establish efficient expression from unintegrated proviral DNA can be rapidly translated to the field of induced pluripotent stem cell (iPS). In addition, the ability to manipulate the short and long-term expression from integrated retroviral vectors has been a major shortcoming for gene transfer. Understanding the role of the IN protein in establishing this epigenetic state is a new and significant area for gene delivery.